Bacillus thuringiensis cryET4 and cryET5 protein insecticidal composition and method of use

ABSTRACT

A Bacillus thuringiensis strain isolate, designated B.t. strain EG5847, exhibits insecticidal activity against lepidopteran insects. Two novel toxin genes from B.t. strain EG5847 designated cryET4 and cryET5 produce insecticidal proteins with activity against a broad spectrum of insects of the order Lepidoptera. The cryET4 gene has a nucleotide base sequence shown in FIG. 1 and listed in SEQ ID NO:1 and produces a CryET4 gene product having the deduced amino acid sequence shown in FIG. 1 and listed in SEQ ID NO:2. The cryET5 gene has a nucleotide base sequence shown in FIG. 2 and listed in SEQ ID NO:3 and produces a CryET5 gene product having the deduced amino acid sequence shown in FIG. 2 and listed in SEQ ID NO:4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.08/176,865, filed Dec. 30, 1993, which is a division of U.S. patentapplication Ser. No. 08/100,709, filed Jul. 29, 1993, now U.S. Pat. No.5,322,687.

FIELD OF THE INVENTION

The present invention relates to lepidopteran-toxic proteins and thegenes coding therefor. In particular, the present invention is directedto genes designated as cryET4 (SEQ ID NO:1) and cryET5 (SEQ ID NO:3) andtheir proteins designated respectively as CryET4 (SEQ ID NO:2) andCryET5 (SEQ ID NO:4).

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (commonly known as B.t.) is a gram-positive soilbacterium that often produces cellular inclusions during sporulationwhich are specifically toxic to certain orders and species of insects.Many different strains of B.t. have been shown to produce theseinclusions of insecticidal crystal protein (ICP). Compositions includingB.t. strains which produce insecticidal proteins have been commerciallyavailable and used as environmentally acceptable insecticides becausethey are quite toxic to the specific target insect, but are harmless toplants and other non-targeted organisms.

B.t. ICP toxins are active in the insect only after ingestion. Afteringestion by an insect, the alkaline pH and proteolytic enzymes in themid-gut solubilize the crystal allowing the release of the toxiccomponents. These toxic components disrupt the mid-gut cells resultingin cessation of feeding and, eventually, death of the insect. B.t. hasproven to be an effective and environmentally safe insecticide indealing with various insect pests.

A number of genes encoding crystal proteins have been cloned from manystrains of B.t. A good overview is set forth in H. Hofte and H. R.Whiteley, Microbiol. Rev., 53, pp. 242-255 (1989), hereinafter "Hofteand Whiteley (1989)." This reference provides a good overview of thegenes and proteins obtained from B.t. and their uses, adopts anomenclature and classification scheme for B.t. genes and proteins, andhas an extensive bibliography.

The nucleotide sequences of ICP genes responsible for a given crystalphenotype and active against the same insect order are generally morerelated, i.e., more homologous, than are the nucleotide sequences ofB.t. genes encoding delta-endotoxin proteins active against differentorders of insects. Hofte and Whiteley (1989) defines an orderedclassification of genes encoding B.t. delta-endotoxin proteins based onhomology of delta-endotoxin amino acid sequences, as well assimilarities in insecticidal activity; a subranking has also beenestablished based upon further refinement of sequence relationship. Asnoted by Hofte and Whiteley (1989), the majority of insecticidal B.t.strains are active against insects of the order Lepidoptera, i.e.,caterpillar insects. Insecticidal crystal proteins specifically activeagainst Lepidoptera have been designated CryI proteins. These ICPs areencoded by cryI genes. Other B.t. strains produce different classes ofcrystal proteins, e.g., CryII proteins are active against lepidopteranand (for CryIIA) dipteran insects; CryIII proteins are insecticidal toinsects of the order Coleoptera, i.e., beetles; and CryIV proteins areactive against insects of the order Diptera, i.e., flies and mosquitoes.A compilation of the amino acid identities for several CryI proteins aswell as CryII, CryIII and CryIV proteins has been determined in Hodgmanand Ellar, J. DNA Sequencing and Mapping, 1, pp. 97-106 (1990).

The CryI family of ICPs contains the largest number of known toxin genesderived from B.t., as evidenced by the survey in Hofte and Whiteley(1989) and by subsequent reports of CryI-type ICPs.

Schnepf et al., J. Biol. Chem., 260, pp. 6264-6272 (1985), reported thecomplete nucleotide sequence for a toxin gene from B.t. kurstaki HD-1.This gene was subsequently classified as cryIA(a) by Hofte and Whiteley(1989). The published open reading frame extends 1176 amino acids andencodes a protein with a calculated molecular mass of 133,500 Daltons(Da). Another gene, also classified as cryIA(a), was isolated from B.t.subsp. kurstaki HD-1 Dipel® by Shibano et al., Gene34, pp. 243-251(1985). As detailed in Table 2 of Hofte and Whiteley (1989), this geneis highly related, especially in the N terminal moiety, to cryIA(a)reported by Schnepf et al. (1985). CryIA(a) protein is broadly activeagainst Lepidoptera; Hofte and Whiteley (1989) reports that four of fivetested lepidopteran insects were sensitive to this toxin.

Other ICP genes subsequently identified as cryIA(a) that are greaterthan 99% identical to the holotype cryIA(a) gene have been identified inB. thuringiensis subspecies aizawai, (Shimizu et al., Agric. Biol.Chem., 52, pp. 1565-1573 (1988)), subspecies kurstaki, (Kondo et al.,Agric. Biol. Chem., 51, pp. 455-463 (1987)), and subspecies entomocidus(Masson et al., Nucleic Acids Res. 17, p. 446 (1989)). The cryI-typenucleotide sequence disclosed in European Patent Application PublicationNo. 0 367 474, published May 9, 1990, of Mycogen Corporation, reveals aDNA sequence related to the cryIA(a) gene and its encoded protein thatis 92% positionally identical to the holotype CryIA(a) ICP.

Wabiko et al., DNA, 5, pp. 305-314 (1986), describe the DNA sequence ofan insecticidal toxin gene from B.t. subsp. berliner1715, subsequentlyclassified as cryIA(b) by Hofte and Whiteley (1989). The molecular massof the protein encoded is 130,615 Da and sequential deletions indicatethat the NH₂ -terminal 612 amino acid polypeptide is toxic tolepidopteran insects. Hofte et al., Eur. J. Biochem., 161, pp. 273-280(1986), describe the cloning and nucleotide sequencing of a variantcrystal protein gene from B.t. subsp. berliner 1715, subsequently alsoclassified as cryIA(b). The cloned gene produces an approximately130,000 Da protein which coincides with the mass of the major proteinobserved in the strain. The gene has an open reading frame of 3465 baseswhich would encode a protein 1155 amino acids in length having a mass of130,533 Da. Similarities of this sequence to the previously reportedsequences for the cloned crystal genes from B.t. kurstakiHD-1, B.t.kurstaki HD-73 and B.t. sotto are discussed in the Hofte et al. (1986)paper. Data identifying a minimal toxic fragment required forinsecticidal activity are also presented. The cryIA(b) gene discussed inHofte et al. (1986) differs in its deduced amino acid sequence by onlytwo amino acids from the CryIA(b) protein reported by wabiko et al.

Other cryIA(b) genes have been disclosed in Geiser et al., Gene, 48, pp.109-118 (1986), Hefford et al., J. Biotechnol., 6, pp. 307-322 (1987),Oeda et al., Gene, 53, pp. 113-119 (1987), Kondo et al., supra,Fischhoff et al., Bio/Technology, 5, pp. 807-813 (1987) and Haider andEllar, Nucl. Acids Res., 16, p. 10927 (1988). Each of these six CryIA(b)ICPs is greater than 99% positionally identical to the holotype CryIA(b)toxin.

Adang et al., Gene, 36, pp. 289-300 (1985), report the cloning andcomplete nucleotide sequence of a crystal protein gene harbored on the75 kilobase (kb) plasmid of strain B.t. subsp. kurstaki HD-73. Therestriction map in the article identified this gene as holotype cryIA(c)under the current classification system of Hofte and Whiteley (1989).The complete sequence of the gene, spanning 3537 nucleotide base pairs(bp), coding for 1178 amino acids and potentially encoding a protein of133,330 Da, is shown in the article. Toxicity data against Manduca sextafor the protein made by the cryIA(c) gene are also presented. CryIA(c)toxins have been isolated from several strains of B.t. subsp. kenyaethat are highly related to the above-noted CryIA(c) toxin from B.t.subsp. kurstaki (greater than 99% positionally identical in deducedamino acid sequence) but whose protein products, although broadly activeagainst lepidopteran insects, nonetheless show quantitatively differenttoxicities for individual insect species (Von Tersch et al., Appl.Environ. Microbiol., 57, pp. 349-358 (1991)).

Brizzard et al., Nucleic Acids Res., 16, pp. 2723-2724 (1988), describethe nucleotide sequence of crystal protein gene cryA4 (subsequentlyclassified as cryIB by Hofte and Whiteley (1989)) isolated from B.t.subsp. thuringiensis HD-2. Hofte and Whiteley (1989) report aninsecticidal specificity of CryIB toxin for Pieris brassicae.

Honee et al., Nucleic Acids Res., 16, p. 6240 (1988), describe thecomplete DNA sequence for the BTVI crystal protein gene isolated fromB.t. subsp. entomocidus 60.5 (holotype cryIC by Hofte and Whiteley(1989)). This protein is reported to exhibit enhanced insecticidalactivities against Spodoptera species.

Sanchis et al., Mol. Microbiol., 3, pp. 229-238 (1989) report thenucleotide sequence for the N-terminal coding region (2470 nucleotides)and 5' flanking region of a gene from B.t. subsp. aizawai7.29 nowclassified as the cryIC gene under the classification system of Hofteand Whiteley (1989). Sanchis et al. disclose similar information aboutthe cryIC gene in European Patent Application Publication No. 0 295 156,published Dec. 14, 1988. The open reading frame encodes a truncatedpolypeptide 824 amino acids long with a calculated mass of 92,906 Da.

A gene isolated from B.t. subspecies aizawai and now classified asholotype cryID under the Hofte and Whiteley (1989) system is disclosedin European Patent Application Publication No. 0 358 557, published Mar.14, 1990 of Plant Genetic Systems, N.V. Hofte and Whiteley (1989) reportselective lepidopteran toxicity against Manduca sexta for the CryIDprotein, the CryID toxin being largely inactive against otherlepidopteran insects tested.

The holotype cryIE gene, found in a B.t. subspecies darmstadiensisstrain, is disclosed in European Patent Application Publication No. 0358 557, supra. A highly related cryIE gene from B.t. subsp. kenyae isdisclosed by Visser et al., J. Bacteriol., 172, pp. 6783-6788 (1990).

Visser, Mol. Gen. Genet., 212, pp. 219-224 (1988) report the isolationand analysis of five toxin genes belonging to four different genefamilies from B.t. entomocidus 60.5, one of which is reported by Honeeet al. (1988), supra. Two of these genes, BTIV and BTVIII, arecryIA(a)-type genes according to the Hofte and Whiteley (1989)classification scheme. The BTVI gene, also reported by Honee et al.(1988) supra, is a cryIC gene according to the Hofte and Whiteley (1989)classification scheme. The authors state that the restriction map foranother gene, designated BTV, closely resembles that identified for thecryID gene isolated from B.t. strain HD68 subsp. aizawai, and disclosedin European Patent Application Publication No. 0 358 557, supra. A fifthgene, designated BTVII, is also identified and its restriction mapdiffers significantly from the other four genes described. Toxicity dataagainst several lepidopteran insects, S. exigua, S. littoralis, H.virescens and P. brassicae, are presented for each of the isolates. TheBTV gene product was inactive against all insects tested. The BTVIprotein is highly active against Spodoptera larvae, and the BTVIIprotein is toxic to P. brassicae.

Additional genes within the cryI family have been reported in theliterature. A gene found in B.t. subsp. aizawai and described as cryIFis disclosed by Chambers et al. in J. Bacteriol., 173, pp. 3966-3976(1991) and in PCT International Publication No. WO91/16434, publishedOct. 31, 1991. A gene described as cryIG from B.t. subsp. galleria isdisclosed by Smulevitch et al., FEBS Lett., 293, pp. 25-28 (1991). Agene that is highly related to the cryIG gene has been isolated fromB.t. DSIR 517 by Gleave et al., J. Gen. Microbiol., 138, pp. 55-62(1992).

A novel gene related to cryI-type genes is disclosed in PCTInternational Publication No. WO 90/13651, published Nov. 15, 1990, ofImperial Chemical Industries PLC. This gene encodes an 81 kDapolypeptide (Cry pJH11) that is broadly insecticidal and more distantlyrelated to the family of cryI sequences than are most other reportedcryI-type sequences. Four cryI-type sequences are disclosed in EuropeanPatent Application Publication No. 0 405 810, published Jan. 2, 1991, ofMycogen Corporation. Inspection of the cryI-type sequences revealed thatone of the disclosed genes (cry 81IB2) belongs to the cryIC class, one(cry 81IB) belongs to the cryID class, and one (cry 81IA) belongs to thecryIF class. The fourth disclosed cryI sequence (cry 81IA2) appears tobelong to a new class. Two cryI sequences are disclosed in EuropeanPatent Application Publication No. 0 401 979, published Dec. 12, 1990,of Mycogen Corporation. One of the disclosed sequences (PS82A2) appearsto encode a novel gene, the other sequence (PS82RR) is highly related tothe novel sequence cry 81IA2 disclosed in European Patent ApplicationPublication No. 0 405 810.

Five novel cry sequences are disclosed in European Patent ApplicationPublication No. 0 462 721, published Dec. 27, 1991, of MycogenCorporation. These Cry proteins are reported to be nematocidal.

SUMMARY OF THE INVENTION

Briefly stated, one aspect of the present invention relates to apurified, isolated cryET4 gene having a nucleotide base sequence codingfor the amino acid sequence shown in FIG. 1 and listed in SEQ ID NO:2.

The isolated cryET4 gene has a coding region extending from nucleotidebases 99 to 3602 (including the stop codon) in the nucleotide basesequence shown in FIG. 1 and listed in SEQ ID NO:1.

The present invention also relates to the isolated CryET4 protein whichis obtainable from the cryET4 gene, and which has the amino acidsequence shown in FIG. 1 (SEQ ID NO:2), and which is insecticidal tolepidopteran insects.

Another aspect of the present invention relates to a purified, isolatedcryET5 gene having a nucleotide base sequence coding for the amino acidsequence shown in FIG. 2 and listed in SEQ ID NO:4.

The isolated cryET5 gene has a coding region extending from nucleotidebases 67 to 3756 (including the stop codon) in the nucleotide basesequence shown in FIG. 2 and listed in SEQ ID NO:3.

The present invention also relates to the isolated CryET5 protein whichis obtainable from the cryET5 gene, and which has the amino acidsequence shown in FIG. 2 (SEQ ID NO:4), and which is insecticidal tolepidopteran insects.

Additionally, the present invention relates to biologically purecultures of a Bacillus thuringiensis bacterium designated as strainEG7279 transformed with a cryET4 gene having a coding region listed inSEQ ID NO:1 and strain EG7283 transformed with a cryET5 gene having acoding region listed in SEQ ID NO:3 or mutants thereof havinginsecticidal activity against lepidopteran insects susceptible to theCryET4 and CryET5 proteins, respectively.

The invention also relates to a biologically pure culture of a Bacillusthuringiensis bacterium designated as strain EG5847 or mutants thereofhaving insecticidal activity against lepidopteran insects susceptible toB.t. strain EG5847. B.t. strain EG5847 is a wild type isolate and is theB.t. strain from which the cryET4 and cryET5 genes were isolated.

Additional aspects of the present invention relate to recombinantplasmids containing the cryET4 and cryET5 genes; bacteria transformedwith the recombinant plasmids and capable of expressing the cryET4and/or cryET5 genes; insecticide compositions comprising the proteinsand/or one or both of the transformed bacteria and/or other bacteriacontaining the CryET4 or CryET5 protein, with an agriculturallyacceptable carrier; a method of controlling lepidopteran insects usingthe insecticides; plants transformed with and capable of expressing thecryET4 and/or cryET5 genes; and hybridization probes containing thecryET4 or cryET5 gene wherein the gene or at least an oligonucleotideportion of it is labeled for such use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises FIGS. 1A through 1J and shows the nucleotide sequenceof the cryET4 gene (SEQ ID NO:1) and the deduced amino acid sequence ofthe CryET4 protein (SEQ ID NO:2).

FIG. 2 comprises FIGS. 2A through 2J and shows the nucleotide sequenceof the cryET5 gene (SEQ ID NO:3) and the deduced amino acid sequence ofthe CryET5 protein (SEQ ID NO:4).

FIG. 3 is a photograph of an ethidium bromide stained agarose gelcontaining size-fractionated plasmids of B.t. strains EG5847 and HD-1,with plasmid sizes in megadaltons (MDa) being shown. The abbreviationsin FIG. 3 are as follows: "ori" indicates the loading site and "lin"means linear DNA.

FIG. 4 is a photograph of a Coomassie blue stained gel containingsize-fractionated proteins from B.t. strains EG5847, EG7279 and EG7283,obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).

FIG. 5 comprises FIGS. 5A and 5B and is a restriction map of therecombinant plasmids pEG291 (5A) and pEGl1108 (5B), both of whichcontain the cloned cryET4 gene. The location and orientation of thecryET4 gene are indicated by the arrow.

FIG. 6 comprises FIGS. 6A, 6B, 6C and 6D and is a restriction map of therecombinant plasmids pEG292 (6A), pEG300 (6B), pEG1110 (6C) and pEG1111(6D). Plasmids pEG292 and pEG300 contain 5' and 3' portions of thecryET5 gene, respectively. Plasmids pEG1110 and pEG1111 contain theentire cloned cryET5 gene. The location and direction of transcriptionof the cryET5 gene are indicated by the arrows.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Two novel Bacillus thuringiensis (B.t.) toxin genes, designated cryET4(SEQ ID NO:1) and cryET5 (SEQ ID NO:3), were obtained from a novel B.t.isolate designated EG5847. Isolation of B.t. strain EG5847, isolation ofthe novel toxin genes cryET4 and cryET5, construction of Bacillus/E.coli shuttle vectors containing cryET4 (pEG1108) and cryET5 (pEG1111),and transformation of pEG1108 and pEG1111 into a B.t. host (B.t. strainEG10368) to produce recombinant B.t. strains EG7279 and EG7283expressing respectively the CryET4 (SEQ ID NO:2) and CryET5 (SEQ IDNO:4) toxin protein gene products, are described generally in theExamples.

Subcultures of B.t. strains EG5847, EG10368, EG7279 and EG7283 weredeposited in the permanent collection of the Agricultural ResearchService Culture Collection, Northern Regional Research Laboratory(NRRL), U.S. Department of Agriculture, 1815 North University Street,Peoria, Ill. 61604, U.S.A. The accession numbers and deposit dates areas follows:

    ______________________________________                                        Subculture    Accession No. Deposit Date                                      ______________________________________                                        B.t. EG5847   NRRL B-21110  June 9, 1993                                      B.t. EG10368  NRRL B-21125  July 20, 1993                                     B.t. EG7279   NRRL B-21112  June 9, 1993                                      B.t. EG7283   NRRL B-21111  June 9, 1993                                      ______________________________________                                    

These microorganism deposits were made under the provisions of the"Budapest Treaty on the International Recognition of the Deposit ofMicroorganism for the Purposes of Patent Procedure." All restrictions onthe availability to the public of these deposited microorganisms will beirrevocably removed upon issuance of a United States patent based onthis application.

The present invention is intended to cover mutants and recombinant orgenetically engineered derivatives, e.g., truncated versions, of thecryET4 gene listed in SEQ ID NO:1 and the cryET5 gene listed in SEQ IDNO:3 that yield a protein with insecticidal properties essentially thesame as those of the CryET4 protein listed in SEQ ID NO:2 and the CryET5protein listed in SEQ ID NO:4. Likewise, the present invention coversthose gene nucleotide base sequences that encode the amino acidsequences of the CryET4 protein (SEQ ID NO:2) and the CryET5 protein(SEQ ID NO:4). Variations may be made in the cryET4 and cryET5 genenucleotide base sequences shown in FIGS. 1 and 2, respectively, andlisted in SEQ ID NO:1 and SEQ ID NO:3, respectively, which do not affectthe amino acid sequence of the gene product, since the degeneracy of thegenetic code is well known to those skilled in the art. Moreover, theremay be some variations or truncations in the coding regions of thecrFET4 and cryET5 nucleotide base sequences which allow expression ofthe gene and production of functionally equivalent forms of the CryET4and CryET5 insecticidal proteins. These variations or truncations, whichcan be determined without undue experimentation by those of ordinaryskill in the art with reference to the present specification, are to beconsidered within the scope of the appended claims, since they are fullyequivalent to the specifically claimed subject matter.

It has been shown that proteins of identical structure and function maybe constructed by changing the amino acid sequence, if such changes donot alter the protein secondary structure (Kaiser and Kezdy, Science,223, pp. 249-255 (1984)). Single amino acid substitutions have beenintroduced by site-directed mutagenesis at various positions of CryIA(a)toxin protein without altering the insecticidal properties of the parenttoxin (Ge et al., Proc. Natl. Acad. Sci. USA, 86, pp. 4037-4041 (1989)).The present invention includes mutants of the amino acid sequencesdisclosed herein which have an unaltered protein secondary structure or,if the structure is altered, where the mutant has retained substantiallyequivalent biological activity compared to the unaltered protein.

The cryET4 gene (SEQ ID NO:1) and cryET5 (SEQ ID NO:3) gene are alsouseful as DNA hybridization probes, for discovering similar or closelyrelated cryET4-type and cryET5-type genes in other B.t. strains. ThecryET4 gene (SEQ ID NO:1) and cryET5 gene (SEQ ID NO:3), or uniqueportions or derivatives thereof capable of hybridizing selectively to atarget nucleic acid, e.g., homologous oligonucleotides of 12 or morenucleotides, or larger portions of the genes, that contain nucleotidesequences unique to the cryET4 gene or cryET5 gene and that aredifferent from similar sized nucleotide segments in known, prior artB.t. toxin genes, can be labeled for use as hybridization probes usingconventional procedures. An exemplary label is a radioactive label.

Both the cryET4 gene (SEQ ID NO:1), its corresponding insecticidalCryET4 protein (SEQ ID NO:2) and the cryET5 gene (SEQ ID NO:2) and itscorresponding insecticidal CryET5 protein (SEQ ID NO:4) were firstidentified in B.t. strain EG5847, a novel B.t. isolate. Thecharacteristics of B.t. strain EG5847 are more fully described in theExamples.

The Bacillus strains described herein may be cultured using conventionalgrowth media and standard fermentation techniques. The B.t. strainsharboring the cryET4 gene (SEQ ID NO:1) or the cryET5 gene (SEQ IDNO:3), or both genes, may be fermented, as described in Example 1, untilthe cultured B.t. cells reach the stage of their growth cycle when theCryET4 crystal protein (SEQ ID NO:2) or the CryET5 crystal protein (SEQID NO:4) is formed. For sporogenous B.t. strains, fermentation istypically continued through the sporulation stage when the crystalprotein is formed along with spores. The B.t. fermentation culture isthen typically harvested by centrifugation, filtration or the like toseparate fermentation culture solids containing the crystal protein fromthe culture medium.

The separated fermentation solids are primarily CryET4 crystal protein(SEQ ID NO:2) or CryET5 crystal protein (SEQ ID NO:4) and B.t. spores(if a sporulating host is employed), along with some cell debris, someintact cells and residual fermentation medium solids. If desired, thecrystal protein may be separated from the other recovered solids viaconventional methods, e.g., density gradient fractionation.

The B.t. strains exemplified in this disclosure are sporulatingvarieties (spore forming or sporogenous strains) but the cryET4 gene(SEQ ID NO:1) and cryET5 gene (SEQ ID NO:3) also have utility inasporogenous Bacillus strains, i.e., strains that produce the crystalprotein without production of spores. It should be understood thatreferences to "fermentation cultures" of B.t. strains containing thecryET4 gene (SEQ ID NO:1) or the cryET5 gene (SEQ ID NO:3) in thisdisclosure are intended to cover sporulated B.t. cultures, i.e., B.t.cultures containing the CryET1 crystal protein and spores, andsporogenous Bacillus strains that have produced crystal proteins duringthe vegetative stage, as well as asporogenous Bacillus strainscontaining the cryET4 gene (SEQ ID NO:1) or cryET5 (SEQ ID NO:3) gene inwhich the culture has reached the growth stage where the crystal proteinis actually produced.

Mutants of B.t. strains harboring the cryET4 gene (SEQ ID NO:1) orcryET5 gene (SEQ ID NO:3) can be made by procedures well known in theart. For example, an asporogenous mutant can be obtained throughethylmethane sulfonate mutagenesis. Mutants can also be made usingultraviolet light and nitrosoguanidine by procedures that are well knownto those skilled in the art. References in this specification to"mutants" of wild-type or recombinant B.t. strains harboring the cryET4gene or cryET5 gene refer to those derivatives which are capable ofproducing toxin protein exhibiting insecticidal activity againstlepidopteran insects, at least equivalent to the insecticidal activityof the parent strain.

The CryET4 protein (SEQ ID NO:2) is an insecticidal compound activeagainst a large number of lepidopteran insects, particularly thosedescribed in Example 4. The CryET4 protein (SEQ ID NO:2) may be used asthe active ingredient in insecticidal formulations useful forcontrolling lepidopteran insects.

The CryET5 protein (SEQ ID NO:4) is an insecticidal compound activeagainst a large number of lepidopteran insects, particularly thosedescribed in Example 4. The CryET5 protein (SEQ ID NO:4) may be used asthe active ingredient in insecticidal formulations useful forcontrolling lepidopteran insects.

Such insecticidal formulations or compositions typically containagriculturally acceptable carriers or adjuvants in addition to theactive ingredient and are prepared and used in a manner well known tothose skilled in the art.

The CryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4) may beemployed in insecticidal formulations in isolated or purified form,e.g., as the crystal protein itself. Alternatively, the CryET4 protein(SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4) may be present in therecovered fermentation solids, obtained from culturing of a Bacillusstrain, e.g., Bacillus thuringiensis or other microorganism hostcarrying the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) andcapable of producing the CryET4 or CryET5 protein. The CryET4 protein orCryET5 protein is thus associated with the B.t. bacterium which producedthe protein, as an intimate mixture of crystal protein, cell debris andspores, if any, in the recovered fermentation solids. The recoveredfermentation solids containing the CryET4 or CryET5 protein may bedried, if desired, prior to incorporation in the insecticidalformulation. Genetically engineered or transformed B.t. strains or otherhost microorganisms containing a recombinant plasmid that expresses thecloned cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3), obtainedby recombinant DNA procedures, may also be used. For construction ofrecombinant B.t. strains containing either the cryET4 gene or cryET5gene, B.t. var. kurstaki strain EG10368 is a preferred host, and thisB.t. strain is utilized in Example 2. B.t. strain EG10368 is acrystal-negative, toxin plasmid-free, naturally occurring mutant of B.t.strain HD73-26 (described in U.S. Pat. No. 5,080,897, issued toGonzalez, Jr. et al. on Jan. 14, 1992) that is highly transformable withrecombinant plasmids, particularly those isolated from E. coli strains,e.g., DH5α.

The formulations or compositions of this invention containing theinsecticidal CryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ IDNO:4) as the active component are applied at an insecticidally effectiveamount which will vary depending on such factors as, for example, thespecific lepidopteran insects to be controlled, the specific plant orcrop to be treated and the method of applying the insecticidally activecompositions.

The insecticide compositions are made by formulating the insecticidallyactive component with the desired agriculturally acceptable carrier. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term "agriculturally acceptable carrier" covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation.

The formulations containing the CryET4 protein (SEQ ID NO:2) or CryET5protein (SEQ ID NO:4) and one or more solid or liquid adjuvants areprepared in known manners, e.g., by homogeneously mixing, blendingand/or grinding the insecticidally active CryET4 or CryET5 proteincomponent with suitable adjuvants using conventional formulationtechniques.

The insecticidal compositions of this invention are applied to theenvironment of the target lepidopteran insect, typically onto thefoliage of the plant or crop to be protected, by conventional methods,preferably by spraying. Other application techniques, e.g., dusting,sprinkling, soaking, soil injection, seed coating, seedling coating orspraying, or the like, are also feasible and may be required for insectsthat cause root or stalk infestation. These application procedures arewell known in the art.

The cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) may beintroduced into a variety of microorganism hosts without undueexperimentation, using procedures well known to those skilled in the artfor transforming suitable hosts under conditions which allow for stablemaintenance and expression of the cloned genes. Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982). Suitable hosts that allow thecryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) gene to beexpressed and the CryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ IDNO:4) to be produced include B.t. and other Bacillus species such as B.subtilis or B. megaterium. A general method for the transformation ofBacillus strains is provided by Macaluso et al. in J. Bacteriol., 173,pp. 1353-1356 (1991) and Mettus et al. in Appl. Environ. Microbiol., 56,pp. 1128-1134 (1990). Genetically altered or engineered microorganismscontaining the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3)can also contain other toxin genes present in the same microorganism;these genes could concurrently produce ICPs different from the CryET4protein or CryET5 protein.

Plant-colonizing or root-colonizing microorganisms may also be employedas the host for the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ IDNO:3). Exemplary microorganism hosts for B.t. toxin genes include theplant-colonizing microbe Clavibacter xyli subsp. cynodontis, asdescribed by Turner et al. in Appl. Environ. Microbiol., 57, pp.3522-3528, and root-colonizing pseudomonad strains, as described byObukowicz et al. in Gene, 45, pp. 327-331 (1986). Procedures such asthose described by Turner et al. (1991) supra, and Obukowicz et al.(1986), supra, are well known to those skilled in the art and availablefor introducing the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ IDNO:3) into such microorganism hosts under conditions which allow forstable maintenance and expression of the gene in the resultingtransformants.

The transformants, i.e., host microorganisms that harbor a cloned genein a recombinant plasmid, can be isolated in accordance withconventional methods, usually employing a selection technique, whichallows growth of only those host microorganisms that contain arecombinant plasmid. The transformants then can be tested forinsecticidal activity. These techniques are standard procedures wellknown to those skilled in the art.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the gene into thehost, availability of expression systems, efficiency of expression,stability of the CryET4 or CryET5 insecticidal protein in the host, andthe presence of auxiliary genetic capabilities. The cellular hostcontaining the insecticidal cryET4 gene (SEQ ID NO:1) or cryET5 gene(SEQ ID NO:3) may be grown in any convenient nutrient medium, whereexpression of the cryET4 gene or cryET5 gene is obtained and CryET4protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4) produced,typically to sporulation. The sporulated cells containing the crystalprotein may then be harvested in accordance with conventional methods,e.g., centrifugation or filtration.

The cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3), particularlythe toxin portion (N-terminal moiety) thereof, may also be incorporatedinto a plant which is capable of expressing the gene and producingCryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4), renderingthe plant more resistant to insect attack. Genetic engineering of plantswith the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) may beaccomplished by introducing the desired DNA containing the gene intoplant tissues or cells, using DNA molecules of a variety of forms andorigins that are well known to those skilled in plant geneticengineering. Examples of techniques for introducing DNA into planttissue are disclosed in European Patent Application Publication No. 0289 479, published Nov. 1, 1988, of Monsanto Company and by Perlak etal. in "Modification of the Coding Sequence Enhances Plant Expression ofInsect Control Protein Genes," Proc. Natl. Acad. Sci. USA, 88, pp.3324-3328 (1991).

DNA containing the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ IDNO:3) or a modified gene, operatively associated with a suitable plantpromoter, e.g., CaMV35S, capable of effecting production of the CryET4protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4), may be deliveredinto the plant cells or tissues directly by infectious plasmids, such asTi, the plasmid from Agrobacterium tumefaciens, viruses ormicroorganisms like A. tumefaciens. Additionally, the use of lysosomesor liposomes, microinjection by mechanical methods and by othertechniques familiar to those skilled in plant genetic engineering may beused.

The basic methods employed in the construction and evaluation of therecombinant plasmids and recombinant microorganism hosts described inthis specification are generally well know to those proficient in theart of molecular cloning. Descriptions of these general laboratoryprocedures and definitions of nomenclature may be found in Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1982) and in a subsequentedition by Sambrook et al. (1989).

The characteristics of the CryET4 protein (SEQ ID NO:2) and CryET5protein (SEQ ID NO:4), sequencing of the cryET4 gene (SEQ ID NO:1) andcryET5 gene (SEQ ID NO:3), comparison of sequence data to known B.t.toxin genes and insecticidal activity of the CryET4 and CryET5 proteinsare described in the following specific, non-limiting examples.

EXAMPLE 1 Characterization of B.t. EG5847

B.t. strain EG5847 is a wild-type isolate, identified by visualexamination of the colony as exhibiting a unique crystal morphology, andwas isolated as a colony from maize dust. The colony containedendospores and bipyramidal and flat, diamond-shaped crystallineinclusions. Subsequent insect bioassay of this wild-type B.t. strainconfirmed its insecticidal activity towards lepidopteran insects.

The complement of native plasmids contained within isolated B.t. EG5847was determined by modified Eckhardt agarose gel electrophoresis asdescribed by Gonzalez, Jr. er al., in Proc. Natl. Acad. Sci. USA, 79,pp. 6951-6955 (1982). The results, as shown in FIG. 3, revealed thepresence of 5, 8, 12, 18 and 110 MDa plasmids. This pattern of nativeplasmids did not correspond to patterns typical of known serovars(Carlton and Gonzalez, pp. 246-252, in Molecular Biology of MicrobialDifferentiation, J. A. Hoch and P. Setlow, ed., American Society forMicrobiology, Washington, D.C. (1985)).

Wild-type B.t. strain EG5847 was grown for five days at 25° C. in DSMmedium (described by Donovan et al. in Appl. Environm. Microbiol., 58,pp. 3921-3927 (1992)) until sporulation and cell lysis had occurred.Recombinant B.t. strains EG7279 (Example 2), containing the cryET4 gene,and EG7283 (Example 2), containing the cryET5 gene, were grown in DSMmedium containing 3 μg of chloramphenicol per ml in a similar manner.Fermentation solids containing spores and crystal proteins were isolatedby centrifugation. Crystal proteins were purified from the spores andcell debris by sucrose density gradient centrifugation (described byKoller et al. in Biochem. Biophys. Res. Communic., 184, pp. 692-699(1992)). Aliquots of the washed crystals were solubilized by heating inLaemmli buffer (10% (w/w) glycerol, 5% (w/w) 2-mercaptoethanol, 1% (w/v)SDS, 0.188M Tris HCl pH 6.8, 0.01% (v/v) bromphenol blue) at 100° C. for5 minutes. The solubilized crystal proteins were size fractionated bySDS-polyacrylamide gel electrophoresis. After size fractionation, theproteins were visualized by staining with Coomassie Blue R-250 dye.

FIG. 4 shows the results of these protein size fractionation analyseswhere lane 1 is a molecular mass size standard, lane 2 is B.t. strainEG5847, lane 3 is B.t. strain EG2729 and lane 4 is B.t. strain EG7283.The numbers on the left side indicate the apparent molecular masses, inkilodaltons (kDa), of the crystal proteins synthesized by the B.t.strains. As shown in lane 3, a crystal protein having a mass ofapproximately 130 kDa was observed from EG7279. As shown in lane 4 forEG7283, a crystal protein having a mass of approximately 130 kDa wasproduced. The wild type strain EG5847 exhibited a large protein band ofapproximately 130 kDa. The observed masses of the crystal proteins arein agreement with the masses predicted from the DNA sequences obtainedin Example 3.

EXAMPLE 2 Cloning of the cryET4 and cryET5 Genes

Genomic DNA was isolated from B.t. strain EG5847 and then digested withHindIII. cryI-like genes were identified by Southern blotting (describedby Southern, J. Mol. Biol. 98, pp. 503-517 (1975)). A radiolabelled 700bp EcoR1 fragment of the cryIA(a) gene (described by Schnepf et al., J.Biol. Chem., 260, pp. 6264-6272 (1985)) was used as a hybridizationprobe to identify cryI-like genes containing HindIII restrictionfragments of EG5847 DNA. The 700 bp cryIA(a) fragment hybridized toseveral HindIII restriction fragments of EG5847 DNA including fragmentsof approximately 5.0 kb and 4.7 kb.

The 5.0 kb and 4.7 kb cryIA(a)-hybridizing HindIII restriction fragmentsof B.t. strain EG5847 were cloned as follows. DNA fragments ofapproximately 4-8 kb from HindIII digests of EG5847 genomic DNA werepurified by agarose gel electrophoresis and electroelution. Thesefragments were ligated to the E. coli plasmid vector pUC18 and theligation mixture was then used to transform E. coli.Ampicillin-resistant E. coli colonies were blotted to nitrocellulosefilters (Grunstein et al., Proc. Natl. Acad. Sci. USA72, pp. 3961-3965(1975)). The filters were probed with the radiolabelled 700 bp cryIA(a)gene fragment. Two positive colonies, designated as E. coli strainsEG7286 and EG7287, were identified and were selected for furtheranalysis.

HindIII digestion of a plasmid, designated pEG291, isolated from E. colistrain EG7286 revealed a HindIII insert fragment 5.0 kb in size inpUC18. The restriction map of plasmid pEG291 is shown in FIG. 5A.

An E. coli/B. thuringiensis shuttle vector containing the 5.0 kb HindIIIfragment was constructed by ligating BamHI digested Bacillus plasmidpNN101 (Norton et al., Plasmid, 13, pp. 211-214 (1985)) into the uniqueBamHI site of pEG291 (FIG. 5B). The resulting plasmid, designatedpEG1108 (FIG. 5B), contains a full length open reading frame which hasbeen designated as the cryET4 gene (SEQ ID NO:1).

E. coli strain EG7287 contained a plasmid, designated pEG292, which hada 4.7 kb HindIII insert in pUC18. DNA sequencing as described in Example3 indicated that an open reading frame present in the 4.7 kb insert wastruncated at its 3'-end (FIG. 6A). The truncated portion was isolatedusing a synthetic oligonucleotide having the sequence5'-AAGTTTCGCATCCATCGATG-3' (SEQ ID NO:5). The oligonucleotide,designated WD162, was homologous to nucleotides 2253 to 2272 of the openreading frame identified in plasmid pEG292. Southern blot analyses asdescribed above indicated that WD162 (SEQ ID NO:5) hybridized to a 3.2kb HincII restriction fragment of EG5847 DNA. Radiolabelled WD162 wasthen used in colony blot experiments as described above to probe E. colicells that contained a plasmid library consisting of size-selectedHincII restriction fragments of EG5847 DNA. Several E. coli colonieshybridized with WD162 and one colony, designated E. coli strain EG7288,was selected for further analysis.

HincII restriction analysis of a recombinant plasmid, designated pEG300,isolated from E. coli EG7288, indicated that a 3.9 kb HincII fragmentwas present in pUC18. DNA sequencing as described below showed thatpEG300 contained an open reading frame truncated at its 5'-end by theHincII cleavage site (FIG. 6B).

The full length open reading frame was constructed by excising a 2.6 kbXbaI-BsmI fragment containing the 5' portion of the open reading framefrom plasmid pEG292 and inserting the fragment into the XbaI-BsmIrestriction sites of plasmid pEG300 (FIGS. 6A and 6B). The resultingplasmid, designated pEG1110 (FIG. 6C), contains a full length openreading frame which has been designated as the cryET5 gene (SEQ IDNO:3).

An E. coli/B. Churingiensis expression vector containing the full lengthopen reading frame of the cryET5 gene was constructed by ligating XbaIdigested Bacillus plasmid pNN101 (Norton, supra) into the unique XbaIsite of plasmid pEG1110 (FIG. 6D). The resulting construct wasdesignated plasmid pEG1111.

Plasmids pEG1108 and pEG1111 are capable of replicating in both E. coliand B.t. The plasmids were transformed by electroporation (Macaluso etal., J. Bacteriol., 173, pp. 1353-1356 (1991)) into the acrystalliferousB.t. strain EG10368 resulting in B.t. strains EG7279(pEG1108) andEG7283(pEG1111), respectively containing the cryET4 and cryET5 genes.Both of these B.t. strains are capable of expressing their respectiveprotein toxin genes, as described in Example 4.

EXAMPLE 3 Sequencing of the cryET4 and cryET5 Genes

The complete DNA sequence of the cryET4 gene was determined usingplasmid pEG291 (Example 2). Plasmid pEG291 was sequenced by standardmethods (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, pp. 5463-5467(1977)). The DNA sequences of the appropriate subclones of the 5.0 kbHindIII fragment were joined to give a continuous sequence of 3713nucleotides which is shown in FIG. 1 and is designated as SEQ ID NO:1.Inspection of the sequence revealed an open reading frame beginning atposition 99 and extending to position 3602 (including the stop codon).The gene has been designated cryET4. The deduced 1167 amino acidsequence of the gene product is shown in FIG. 1 and is designated as SEQID NO:2. The mass of the CryET4 protein (SEQ ID NO:2) encoded by thecryET4 gene (SEQ ID NO:1), as deduced from the open reading frame, is132,774 Da. Among CryI-type protein toxins reported in the literature,the CryIA(a) protein appears to be most closely related to the CryET4protein. CryET4 exhibits 69% amino acid homology with CryIA(a).

The complete DNA sequence of the cryET5 gene (SEQ ID NO:3) wasdetermined by the Sanger method as described above. Subcloned genefragments from pEG292, pEG300 and pEG1110 were sequenced. The DNAsequences of the subcloned fragments were joined to give a continuoussequence of 3,934 nucleotides which is shown in FIG. 2 and is designatedas SEQ ID NO:3. Inspection of the sequence revealed an open readingframe beginning at position 67 and extending to position 3756 (includingthe stop codon). The gene has been designated cryET5. The deduced 1229amino acid sequence of the gene product encoded by the cryET5 gene (SEQID NO:3) is shown in FIG. 2 and is designated as SEQ ID NO:4. The massof the CryET5 protein (SEQ ID NO:4) encoded by the cryET5 gene (SEQ IDNO:3), as deduced from the open reading frame, is 139,783 Da. AmongCryI-type proteins reported in the literature, the CryIB protein appearsto be most closely related to the CryET5 protein. CryET5 exhibits 83%amino acid homology with CryIB.

EXAMPLE 4 Insecticidal Activity of Recombinant Strains Harboring cryET4and cryET5 Genes

PLC₅₀ values of purified CryET4 and CryET5 crystal proteins weredetermined against lepidopteran insects, and these are listed in Tables1 and 2, respectively. The PLC₅₀ dose is that amount of insecticidalcrystal protein (ICP) which killed half of the insects tested, i.e., themedian lethal concentration. CryET4 and CryET5 crystal proteins wereisolated from B.t. strains EG7279 and EG7283, respectively, (describedin Example 2) by sucrose density gradient centrifugation as describedabove. The amount of crystal protein recovered from the gradients wasquantified by the Bradford protein assay (Bradford, Anal. Biochem., 72,p. 248 (1976)) after solubilization of the recovered crystal proteinswith base and a reducing agent. Known amounts of purified crystals werediluted in 0.005% Triton® X-100 (v/v). Aliquots of eight two-fold serialdilutions (50 μl ) were applied to the surfaces of 32 wells (1.8 cm²surface area) containing insect diet and dried for 1 hour at 30° C. Ageneral purpose Noctuidae artificial diet (E.G. King et al., Handbook ofInsect Rearing, Vol. 2, P. Singh and R. F. Moore (eds.), pp. 323-328,Elsevier Science Publishers B.V., Amsterdam (1985)) was used forTrichoplusia ni, Ostrinia nubilalis and Heliothis virescens. Otherstandard diets were used for the other lepidopteran insects tested. Oneneonate larva (third-instar larva in the case of P. xylostella) wasadded to each well, and the wells were incubated at 30° C. Mortality wasrecorded after seven days.

The insecticidal activity of CryET4 protein was compared with theactivity of CryIA(a) protein (Schnepf et al., J. Biol. Chem., 260, pp.6264-6272 (1985)). CryET4 exhibits a 69% amino acid sequence homologywith CryIA(a). The results are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        PLC.sub.50 Bioassay Activity of Purified CryET4                                          PLC.sub.50 (ng ICP/well)                                           Insect Species                                                                             CryET4         CryIA(a)                                          ______________________________________                                        Heliothis virescens                                                                        593 (493-711)**                                                                              94 (76-113)                                       Helicoverpa zea                                                                            1,290 (1,046-1,599)                                                                          3,725 (3,004-4,551)                               Lymantria dispar                                                                           9,929 (5,767-26,039)                                                                         185 (138-243)                                     Ostrinia nubilalis                                                                         197 (121-299)  34 (27-42)                                        Pseudoplusia includens                                                                     33 (29-37)     14 (12-16)                                        Plutella xylostella                                                                        30 (22-41)     12 (10-14)                                        Javelin ®*-resistant                                                                   4,758 (3,135-6,897)                                                                          >50,000                                           P. xylostella                                                                 Spodoptera exiqua                                                                          1,748 (1,286-2,591)                                                                          >20,000                                           Spodoptera frugiperda                                                                      1,161 (555-2,115)                                                                            >10,000                                           Trichoplusia ni                                                                            62 (53-74)     80 (54-113)                                       ______________________________________                                         *Javelin is a commercial B.t. bioinsecticide.                                 **Range in parentheses indicates 95% confidence level.                   

The PLC₅₀ results in Table 1 indicate that the CryET4 protein (SEQ IDNO:2) exhibits good insecticidal activity to a broad spectrum oflepidopteran insects.

The results show that the CryET4 protein is more toxic than CryIA(a)against Helicoverpa zea (corn earworm/bollworm), Javelin®-resistantPlutella xylostella (diamondback moth), Spodoptera exigua (beetarmyworm) and Spodoptera frugiperda (fall armyworm).

Particularly noteworthy is the very good activity against Spodopteraexigua (beet armyworm), an insect pest that not only is not susceptibleto CryIA(a), but also is recalcitrant to most B.t. toxin proteins, andvery good activity against Spodoptera frugiperda (fall armyworm),another recalcitrant insect pest. Activity against Pseudoplusiaincludens (soybean looper), Plutella xylostella (diamondback moth) andTrichoplusia ni (cabbage looper) was also good, comparable to thatexhibited by CryIA(a).

Insect bioassay tests with CryET4 protein were also carried out againstanother lepidopteran insect, Agrotis ipsilon (black cutworm), which wasfound not to be susceptible to control with CryET4.

The insecticidal activity of CryET5 protein was compared with theactivity of CryIB protein (Brizzard et al., Nucleic Acids Res. 16,2723-2724 (1988)). CryET5 exhibits an 83% amino acid sequence homologywith CryIB. Dilutions of purified CryET5 crystals were prepared in0.005% Triton® X-100. Aliquots of appropriate dilutions (50 μl) wereapplied to the surfaces of 32 wells and assayed as indicated above. Theresults are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        PLC.sub.50 Bioassay Activity of Purified CryET5                                          PLC.sub.50 (ng ICP/well)                                           Insect Species                                                                             CryET5        CryIB                                              ______________________________________                                        Lymantria dispar                                                                           880 (555-1,397)**                                                                           3,580 (1,293-20,123)                               Ostrinia nubilalis                                                                         32 (29-37)    83 (51-123)                                        Pseudoplusia includens                                                                     555 (437-646) 52 (44-61)                                         Plutella xylostella                                                                        157 (127-193) 27 (23-32)                                         Javelin ®*-resistant                                                                   47 (23-80)    43 (35-55)                                         P. xylostella                                                                 Spodoptera frugiperda                                                                      2,812 (1,831-4,514)                                                                         >10,000                                            Trichoplusia ni                                                                            22 (19-27)    205 (176-241)                                      ______________________________________                                         *Javelin is a commercial B.t. bioinsecticide.                                 **Range in parentheses indicates 95% confidence level.                   

The PLC₅₀ results in Table 2 indicate that the CryET5 protein (SEQ IDNO:4) exhibits good insecticidal activity to a broad spectrum oflepidopteran insects.

The results show that the CryET5 protein is more toxic than CryIBagainst Spodoptera frugiperda (fall armyworm) and Trichoplusia ni(cabbage looper). The CryET5 protein and CryIB protein both exhibitedexcellent insecticidal activity against Javelin®-resistant Plutellaxylostella (diamondback moth), a B.t. -resistant insect pest that is notsusceptible to CryIA-type toxin proteins, and to Ostrinia nubilalis(European corn borer).

Insect bioassay tests with CryET5 protein were also carried out againsta few other lepidopteran insects, but these were found not to besusceptible to control with CryET5: Agrotis ipsilon (black cutworm),Heliothis virescens (tobacco budworm), Helicoverpa zea (cornearworm/bollworm) and Spodoptera exigua (beet armyworm).

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification as indicating the scope of theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3713 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 99..3602                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAATTCATAATATGAATCATACGTTTTAAAGTGTTGTGAAGAAAAGAGAATTGATCTTTA60                GAATTTTTTTATTTTAACCAAAGAGAAAGGGGTAACTTATGGAGATAAATAAT113                      MetGluIleAsnAsn                                                               15                                                                            CAGAAGCAATGCATACCATATAATTGCTTAAGTAATCCTGAGGAAGTA161                           GlnLysGlnCysIleProTyrAsnCysLeuSerAsnProGluGluVal                              101520                                                                        CTTTTGGATGGGGAGAGGATATTACCTGATATCGATCCACTCGAAGTT209                           LeuLeuAspGlyGluArgIleLeuProAspIleAspProLeuGluVal                              253035                                                                        TCTTTGTCGCTTTTGCAATTTCTTTTGAATAACTTTGTTCCAGGGGGA257                           SerLeuSerLeuLeuGlnPheLeuLeuAsnAsnPheValProGlyGly                              404550                                                                        GGCTTTATTTCAGGATTAGTTGATAAAATATGGGGGGCTTTGAGACCA305                           GlyPheIleSerGlyLeuValAspLysIleTrpGlyAlaLeuArgPro                              556065                                                                        TCTGAATGGGACTTATTTCTTGCACAGATTGAACGGTTGATTGATCAA353                           SerGluTrpAspLeuPheLeuAlaGlnIleGluArgLeuIleAspGln                              70758085                                                                      AGAATAGAAGCAACAGTAAGAGCAAAAGCAATCACTGAATTAGAAGGA401                           ArgIleGluAlaThrValArgAlaLysAlaIleThrGluLeuGluGly                              9095100                                                                       TTAGGGAGAAATTATCAAATATACGCTGAAGCATTTAAAGAATGGGAA449                           LeuGlyArgAsnTyrGlnIleTyrAlaGluAlaPheLysGluTrpGlu                              105110115                                                                     TCAGATCCTGATAACGAAGCGGCTAAAAGTAGAGTAATTGATCGCTTT497                           SerAspProAspAsnGluAlaAlaLysSerArgValIleAspArgPhe                              120125130                                                                     CGTATACTTGATGGTCTAATTGAAGCAAATATCCCTTCATTTCGGATA545                           ArgIleLeuAspGlyLeuIleGluAlaAsnIleProSerPheArgIle                              135140145                                                                     ATTGGATTTGAAGTGCCACTTTTATCGGTTTATGTTCAAGCAGCTAAT593                           IleGlyPheGluValProLeuLeuSerValTyrValGlnAlaAlaAsn                              150155160165                                                                  CTACATCTCGCTCTATTGAGAGATTCTGTTATTTTTGGAGAGAGATGG641                           LeuHisLeuAlaLeuLeuArgAspSerValIlePheGlyGluArgTrp                              170175180                                                                     GGATTGACGACAAAAAATGTCAATGATATCTATAATAGACAAATTAGA689                           GlyLeuThrThrLysAsnValAsnAspIleTyrAsnArgGlnIleArg                              185190195                                                                     GAAATTCATGAATATAGCAATCATTGCGTAGATACGTATAACACAGAA737                           GluIleHisGluTyrSerAsnHisCysValAspThrTyrAsnThrGlu                              200205210                                                                     CTAGAACGTCTAGGGTTTAGATCTATAGCGCAGTGGAGAATATATAAT785                           LeuGluArgLeuGlyPheArgSerIleAlaGlnTrpArgIleTyrAsn                              215220225                                                                     CAGTTTAGAAGAGAACTAACACTAACTGTATTAGATATTGTCGCTCTT833                           GlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeu                              230235240245                                                                  TTCCCGAACTATGACAGTAGACTGTATCCGATCCAAACTTTTTCTCAA881                           PheProAsnTyrAspSerArgLeuTyrProIleGlnThrPheSerGln                              250255260                                                                     TTGACAAGAGAAATTGTTACATCCCCAGTAAGCGAATTTTATTATGGT929                           LeuThrArgGluIleValThrSerProValSerGluPheTyrTyrGly                              265270275                                                                     GTTATTAATAGTGGTAATATAATTGGTACTCTTACTGAACAGCAGATA977                           ValIleAsnSerGlyAsnIleIleGlyThrLeuThrGluGlnGlnIle                              280285290                                                                     AGGCGACCACATCTTATGGACTTCTTTAACTCCATGATCATGTATACA1025                          ArgArgProHisLeuMetAspPhePheAsnSerMetIleMetTyrThr                              295300305                                                                     TCAGATAATAGACGGGAACATTATTGGTCAGGACTTGAAATGACGGCT1073                          SerAspAsnArgArgGluHisTyrTrpSerGlyLeuGluMetThrAla                              310315320325                                                                  TATTTTACAGGATTTGCAGGAGCTCAAGTGTCATTCCCTTTAGTCGGG1121                          TyrPheThrGlyPheAlaGlyAlaGlnValSerPheProLeuValGly                              330335340                                                                     ACTAGAGGGGAGTCAGCTCCACCATTAACTGTTAGAAGTGTTAATGAT1169                          ThrArgGlyGluSerAlaProProLeuThrValArgSerValAsnAsp                              345350355                                                                     GGAATTTATAGAATATTATCGGCACCGTTTTATTCAGCGCCTTTTCTA1217                          GlyIleTyrArgIleLeuSerAlaProPheTyrSerAlaProPheLeu                              360365370                                                                     GGCACCATTGTATTGGGAAGTCGTGGAGAAAAATTTGATTTTGCGCTT1265                          GlyThrIleValLeuGlySerArgGlyGluLysPheAspPheAlaLeu                              375380385                                                                     AATAATATTTCACCTCCGCCATCTACAATATACAGACATCCTGGAACA1313                          AsnAsnIleSerProProProSerThrIleTyrArgHisProGlyThr                              390395400405                                                                  GTAGATTCACTAGTCAGTATACCGCCACAGGATAATAGCGTACCACCG1361                          ValAspSerLeuValSerIleProProGlnAspAsnSerValProPro                              410415420                                                                     CACAGGGGATCTAGTCATCGATTAAGTCATGTTACAATGCGCGCAAGT1409                          HisArgGlySerSerHisArgLeuSerHisValThrMetArgAlaSer                              425430435                                                                     TCCCCTATATTCCATTGGACGCATCGCAGCGCAACCACTACAAATACA1457                          SerProIlePheHisTrpThrHisArgSerAlaThrThrThrAsnThr                              440445450                                                                     ATTAATCCAAATGCTATTATCCAAATACCACTAGTAAAAGCATTTAAC1505                          IleAsnProAsnAlaIleIleGlnIleProLeuValLysAlaPheAsn                              455460465                                                                     CTTCATTCAGGTGCCACTGTTGTTAGAGGACCAGGGTTTACAGGTGGT1553                          LeuHisSerGlyAlaThrValValArgGlyProGlyPheThrGlyGly                              470475480485                                                                  GATATCCTTCGAAGAACGAATACTGGCACATTTGCAGATATGAGAGTA1601                          AspIleLeuArgArgThrAsnThrGlyThrPheAlaAspMetArgVal                              490495500                                                                     AATATTACTGGGCCATTATCCCAAAGATATCGTGTAAGAATTCGCTAT1649                          AsnIleThrGlyProLeuSerGlnArgTyrArgValArgIleArgTyr                              505510515                                                                     GCTTCTACGACAGATTTACAATTTTTCACGAGAATCAATGGAACTTCT1697                          AlaSerThrThrAspLeuGlnPhePheThrArgIleAsnGlyThrSer                              520525530                                                                     GTAAATCAAGGTAATTTCCAAAGAACTATGAATAGAGGGGATAATTTA1745                          ValAsnGlnGlyAsnPheGlnArgThrMetAsnArgGlyAspAsnLeu                              535540545                                                                     GAATCTGGAAACTTTAGGACTGCAGGATTTAGTACGCCTTTTAGTTTT1793                          GluSerGlyAsnPheArgThrAlaGlyPheSerThrProPheSerPhe                              550555560565                                                                  TCAAATGCGCAAAGTACATTCACATTGGGTACTCAGGCTTTTTCAAAT1841                          SerAsnAlaGlnSerThrPheThrLeuGlyThrGlnAlaPheSerAsn                              570575580                                                                     CAGGAAGTTTATATAGATCGAATTGAATTTGTCCCGGCAGAAGTAACA1889                          GlnGluValTyrIleAspArgIleGluPheValProAlaGluValThr                              585590595                                                                     TTCGAGGCAGAATCTGATTTAGAAAGAGCGCAAAAGGCGGTGAATGCC1937                          PheGluAlaGluSerAspLeuGluArgAlaGlnLysAlaValAsnAla                              600605610                                                                     CTGTTTACTTCTACAAACCAACTAGGGCTAAAAACAGATGTGACGGAT1985                          LeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAspValThrAsp                              615620625                                                                     TATCAGATTGATCAAGTGTCCAATTTAGTAGAATGTTTATCAGATGAA2033                          TyrGlnIleAspGlnValSerAsnLeuValGluCysLeuSerAspGlu                              630635640645                                                                  TTTTGTCTGGATGAAAAGAGAGAATTGTCCGAGAAAGTCAAACATGCA2081                          PheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAla                              650655660                                                                     AAGCGACTTAGTGATAAGCGGAACCTACTTCAAGATCCAAACTTCACA2129                          LysArgLeuSerAspLysArgAsnLeuLeuGlnAspProAsnPheThr                              665670675                                                                     TCTATCAATAGACAACTAGACCGTGGATGGAGAGGAAGTACGGATATT2177                          SerIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIle                              680685690                                                                     ACCATCCAAGGAGGAAATGACGTATTCAAAGAGAATTACGTCACACTA2225                          ThrIleGlnGlyGlyAsnAspValPheLysGluAsnTyrValThrLeu                              695700705                                                                     CCAGGTACCTTTGATGAGTGTTATCCAACGTATTTGTATCAAAAAATA2273                          ProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIle                              710715720725                                                                  GATGAGTCAAAATTAAAAGCCTATACTCGCTATGAATTAAGAGGGTAT2321                          AspGluSerLysLeuLysAlaTyrThrArgTyrGluLeuArgGlyTyr                              730735740                                                                     ATTGAAGATAGTCAAGATTTAGAAGTCTATTTGATTCGTTACAATGCG2369                          IleGluAspSerGlnAspLeuGluValTyrLeuIleArgTyrAsnAla                              745750755                                                                     AAACATGAAACAGTAAATGTTCCCGGTACAGGGTCCTTATGGCCGCTT2417                          LysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeu                              760765770                                                                     TCAGTCGAAAGCCCAATCGGAAGGTGCGGAGAACCGAATCGATGTGTG2465                          SerValGluSerProIleGlyArgCysGlyGluProAsnArgCysVal                              775780785                                                                     CCACATATTGAATGGAATCCTGATTTAGATTGTTCGTGTAGGGATGGG2513                          ProHisIleGluTrpAsnProAspLeuAspCysSerCysArgAspGly                              790795800805                                                                  GAGAAGTGTGCCCATCATTCGCATCATTTCTCTCTAGATATTGATGTT2561                          GluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspVal                              810815820                                                                     GGATGTACAGACCTAAATGAGGACCTAGGTGTATGGGTGATCTTTAAG2609                          GlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLys                              825830835                                                                     ATTAAAACGCAGGATGGCCATGCAAGATTAGGAAATCTAGAGTTTCTC2657                          IleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeu                              840845850                                                                     GAAGAGAAACCATTGTTAGGAGAAGCGTTAGCTCGTGTGAAAAGAGCG2705                          GluGluLysProLeuLeuGlyGluAlaLeuAlaArgValLysArgAla                              855860865                                                                     GAGAAAAAATGGAGAGACAAACGCGAACAATTGCAGTTTGAAACGAAT2753                          GluLysLysTrpArgAspLysArgGluGlnLeuGlnPheGluThrAsn                              870875880885                                                                  ATCGTTTACAAAGAGGCAAAAGAATCTGTAGATGCTTTATTCGTAGAT2801                          IleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsp                              890895900                                                                     TCTCACTATAATAGATTACAAGCGGATACGAACATTACGATGATTCAT2849                          SerHisTyrAsnArgLeuGlnAlaAspThrAsnIleThrMetIleHis                              905910915                                                                     GCGGCAGATAAACGCGTTCATCGAATCCGAGAGGCTTATCTTCCGGAA2897                          AlaAlaAspLysArgValHisArgIleArgGluAlaTyrLeuProGlu                              920925930                                                                     TTATCCGTTATCCCAGGTGTAAATGCGGACATTTTTGAAGAATTAGAA2945                          LeuSerValIleProGlyValAsnAlaAspIlePheGluGluLeuGlu                              935940945                                                                     GGTCTTATTTTCACTGCATTCTCCCTATATGATGCGAGAAATATCATT2993                          GlyLeuIlePheThrAlaPheSerLeuTyrAspAlaArgAsnIleIle                              950955960965                                                                  AAAAACGGTGATTTCAATAATGGTTTATCGTGTTGGAACGTGAAAGGG3041                          LysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGly                              970975980                                                                     CATGTAGATATACAACAGAATGATCATCGTTCTGTCCTCGTTGTCCCG3089                          HisValAspIleGlnGlnAsnAspHisArgSerValLeuValValPro                              985990995                                                                     GAATGGGAATCAGAGGTATCACAAGAAGTCCGCGTATGTCCAGGTCGT3137                          GluTrpGluSerGluValSerGlnGluValArgValCysProGlyArg                              100010051010                                                                  GGCTATATTCTTCGTGTCACAGCGTACAAAGAGGGCTACGGAGAAGGA3185                          GlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGly                              101510201025                                                                  TGCGTAACGATCCATGAGATCGAAGACAATACAGACGAATTGAAGTTT3233                          CysValThrIleHisGluIleGluAspAsnThrAspGluLeuLysPhe                              1030103510401045                                                              AGTAACTGCATAGAAGAGGAAGTCTATCCAACGGATACAGGTAATGAT3281                          SerAsnCysIleGluGluGluValTyrProThrAspThrGlyAsnAsp                              105010551060                                                                  TATACTGCACACCAAGGTACAACAGGATGCGCAGATGCATGTAATTCC3329                          TyrThrAlaHisGlnGlyThrThrGlyCysAlaAspAlaCysAsnSer                              106510701075                                                                  CGTAATGTTGGATATGAGGATGGATATGAAATAAATACTACAGCATCT3377                          ArgAsnValGlyTyrGluAspGlyTyrGluIleAsnThrThrAlaSer                              108010851090                                                                  GTTAATTACAAACCGACTTATGAAGAAGAAATGTATACAGATGTACGA3425                          ValAsnTyrLysProThrTyrGluGluGluMetTyrThrAspValArg                              109511001105                                                                  AGAGATAATCATTGTGAATATGACAGAGGATATGGGAACCATACACCG3473                          ArgAspAsnHisCysGluTyrAspArgGlyTyrGlyAsnHisThrPro                              1110111511201125                                                              TTACCAGCTGGTTATGTAACAAAAGAATTAGAGTACTTCCCTGAAACA3521                          LeuProAlaGlyTyrValThrLysGluLeuGluTyrPheProGluThr                              113011351140                                                                  GATACAGTATGGATAGAGATTGGAGAAACGGAAGGAACATTCATCGTA3569                          AspThrValTrpIleGluIleGlyGluThrGluGlyThrPheIleVal                              114511501155                                                                  GATAGTGTGGAATTACTCCTCATGGAGGAATAAGATTGTACGAAATCGAC3619                        AspSerValGluLeuLeuLeuMetGluGlu                                                11601165                                                                      TTTAAATGGCTCATTCTAAACAAAAAGTAGTCGTCTAATCTCTGTAACAAATAGAAAAGT3679              AAATATTTGTAGAAAAAAGAAAAAGGACATTACT3713                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1167 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluIleAsnAsnGlnLysGlnCysIleProTyrAsnCysLeuSer                              151015                                                                        AsnProGluGluValLeuLeuAspGlyGluArgIleLeuProAspIle                              202530                                                                        AspProLeuGluValSerLeuSerLeuLeuGlnPheLeuLeuAsnAsn                              354045                                                                        PheValProGlyGlyGlyPheIleSerGlyLeuValAspLysIleTrp                              505560                                                                        GlyAlaLeuArgProSerGluTrpAspLeuPheLeuAlaGlnIleGlu                              65707580                                                                      ArgLeuIleAspGlnArgIleGluAlaThrValArgAlaLysAlaIle                              859095                                                                        ThrGluLeuGluGlyLeuGlyArgAsnTyrGlnIleTyrAlaGluAla                              100105110                                                                     PheLysGluTrpGluSerAspProAspAsnGluAlaAlaLysSerArg                              115120125                                                                     ValIleAspArgPheArgIleLeuAspGlyLeuIleGluAlaAsnIle                              130135140                                                                     ProSerPheArgIleIleGlyPheGluValProLeuLeuSerValTyr                              145150155160                                                                  ValGlnAlaAlaAsnLeuHisLeuAlaLeuLeuArgAspSerValIle                              165170175                                                                     PheGlyGluArgTrpGlyLeuThrThrLysAsnValAsnAspIleTyr                              180185190                                                                     AsnArgGlnIleArgGluIleHisGluTyrSerAsnHisCysValAsp                              195200205                                                                     ThrTyrAsnThrGluLeuGluArgLeuGlyPheArgSerIleAlaGln                              210215220                                                                     TrpArgIleTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeu                              225230235240                                                                  AspIleValAlaLeuPheProAsnTyrAspSerArgLeuTyrProIle                              245250255                                                                     GlnThrPheSerGlnLeuThrArgGluIleValThrSerProValSer                              260265270                                                                     GluPheTyrTyrGlyValIleAsnSerGlyAsnIleIleGlyThrLeu                              275280285                                                                     ThrGluGlnGlnIleArgArgProHisLeuMetAspPhePheAsnSer                              290295300                                                                     MetIleMetTyrThrSerAspAsnArgArgGluHisTyrTrpSerGly                              305310315320                                                                  LeuGluMetThrAlaTyrPheThrGlyPheAlaGlyAlaGlnValSer                              325330335                                                                     PheProLeuValGlyThrArgGlyGluSerAlaProProLeuThrVal                              340345350                                                                     ArgSerValAsnAspGlyIleTyrArgIleLeuSerAlaProPheTyr                              355360365                                                                     SerAlaProPheLeuGlyThrIleValLeuGlySerArgGlyGluLys                              370375380                                                                     PheAspPheAlaLeuAsnAsnIleSerProProProSerThrIleTyr                              385390395400                                                                  ArgHisProGlyThrValAspSerLeuValSerIleProProGlnAsp                              405410415                                                                     AsnSerValProProHisArgGlySerSerHisArgLeuSerHisVal                              420425430                                                                     ThrMetArgAlaSerSerProIlePheHisTrpThrHisArgSerAla                              435440445                                                                     ThrThrThrAsnThrIleAsnProAsnAlaIleIleGlnIleProLeu                              450455460                                                                     ValLysAlaPheAsnLeuHisSerGlyAlaThrValValArgGlyPro                              465470475480                                                                  GlyPheThrGlyGlyAspIleLeuArgArgThrAsnThrGlyThrPhe                              485490495                                                                     AlaAspMetArgValAsnIleThrGlyProLeuSerGlnArgTyrArg                              500505510                                                                     ValArgIleArgTyrAlaSerThrThrAspLeuGlnPhePheThrArg                              515520525                                                                     IleAsnGlyThrSerValAsnGlnGlyAsnPheGlnArgThrMetAsn                              530535540                                                                     ArgGlyAspAsnLeuGluSerGlyAsnPheArgThrAlaGlyPheSer                              545550555560                                                                  ThrProPheSerPheSerAsnAlaGlnSerThrPheThrLeuGlyThr                              565570575                                                                     GlnAlaPheSerAsnGlnGluValTyrIleAspArgIleGluPheVal                              580585590                                                                     ProAlaGluValThrPheGluAlaGluSerAspLeuGluArgAlaGln                              595600605                                                                     LysAlaValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLys                              610615620                                                                     ThrAspValThrAspTyrGlnIleAspGlnValSerAsnLeuValGlu                              625630635640                                                                  CysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGlu                              645650655                                                                     LysValLysHisAlaLysArgLeuSerAspLysArgAsnLeuLeuGln                              660665670                                                                     AspProAsnPheThrSerIleAsnArgGlnLeuAspArgGlyTrpArg                              675680685                                                                     GlySerThrAspIleThrIleGlnGlyGlyAsnAspValPheLysGlu                              690695700                                                                     AsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyr                              705710715720                                                                  LeuTyrGlnLysIleAspGluSerLysLeuLysAlaTyrThrArgTyr                              725730735                                                                     GluLeuArgGlyTyrIleGluAspSerGlnAspLeuGluValTyrLeu                              740745750                                                                     IleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGly                              755760765                                                                     SerLeuTrpProLeuSerValGluSerProIleGlyArgCysGlyGlu                              770775780                                                                     ProAsnArgCysValProHisIleGluTrpAsnProAspLeuAspCys                              785790795800                                                                  SerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSer                              805810815                                                                     LeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyVal                              820825830                                                                     TrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGly                              835840845                                                                     AsnLeuGluPheLeuGluGluLysProLeuLeuGlyGluAlaLeuAla                              850855860                                                                     ArgValLysArgAlaGluLysLysTrpArgAspLysArgGluGlnLeu                              865870875880                                                                  GlnPheGluThrAsnIleValTyrLysGluAlaLysGluSerValAsp                              885890895                                                                     AlaLeuPheValAspSerHisTyrAsnArgLeuGlnAlaAspThrAsn                              900905910                                                                     IleThrMetIleHisAlaAlaAspLysArgValHisArgIleArgGlu                              915920925                                                                     AlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAspIle                              930935940                                                                     PheGluGluLeuGluGlyLeuIlePheThrAlaPheSerLeuTyrAsp                              945950955960                                                                  AlaArgAsnIleIleLysAsnGlyAspPheAsnAsnGlyLeuSerCys                              965970975                                                                     TrpAsnValLysGlyHisValAspIleGlnGlnAsnAspHisArgSer                              980985990                                                                     ValLeuValValProGluTrpGluSerGluValSerGlnGluValArg                              99510001005                                                                   ValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGlu                              101010151020                                                                  GlyTyrGlyGluGlyCysValThrIleHisGluIleGluAspAsnThr                              1025103010351040                                                              AspGluLeuLysPheSerAsnCysIleGluGluGluValTyrProThr                              104510501055                                                                  AspThrGlyAsnAspTyrThrAlaHisGlnGlyThrThrGlyCysAla                              106010651070                                                                  AspAlaCysAsnSerArgAsnValGlyTyrGluAspGlyTyrGluIle                              107510801085                                                                  AsnThrThrAlaSerValAsnTyrLysProThrTyrGluGluGluMet                              109010951100                                                                  TyrThrAspValArgArgAspAsnHisCysGluTyrAspArgGlyTyr                              1105111011151120                                                              GlyAsnHisThrProLeuProAlaGlyTyrValThrLysGluLeuGlu                              112511301135                                                                  TyrPheProGluThrAspThrValTrpIleGluIleGlyGluThrGlu                              114011451150                                                                  GlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu                                 115511601165                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3934 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 67..3756                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 2253..2272                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AAACTATTCAATGGAGAAAAATTGAATAGTTGTAATGTAAGCACACCGAAAAAAGGAGGA60                GTTATATTGACTTCAAATAGGAAAAATGAGAATGAAATTATAAATGCT108                           LeuThrSerAsnArgLysAsnGluAsnGluIleIleAsnAla                                    1510                                                                          TTATCGATTCCAACGGTATCGAATCCTTCCACGCAAATGAATCTATCA156                           LeuSerIleProThrValSerAsnProSerThrGlnMetAsnLeuSer                              15202530                                                                      CCAGATGCTCGTATTGAAGATAGCTTGTGTGTAGCCGAGGTGAACAAT204                           ProAspAlaArgIleGluAspSerLeuCysValAlaGluValAsnAsn                              354045                                                                        ATTGATCCATTTGTTAGCGCATCAACAGTCCAAACGGGTATAAACATA252                           IleAspProPheValSerAlaSerThrValGlnThrGlyIleAsnIle                              505560                                                                        GCTGGTAGAATATTGGGCGTATTAGGTGTGCCGTTTGCTGGACAACTA300                           AlaGlyArgIleLeuGlyValLeuGlyValProPheAlaGlyGlnLeu                              657075                                                                        GCTAGTTTTTATAGTTTTCTTGTTGGGGAATTATGGCCTAGTGGCAGA348                           AlaSerPheTyrSerPheLeuValGlyGluLeuTrpProSerGlyArg                              808590                                                                        GATCCATGGGAAATTTTCCTGGAACATGTAGAACAACTTATAAGACAA396                           AspProTrpGluIlePheLeuGluHisValGluGlnLeuIleArgGln                              95100105110                                                                   CAAGTAACAGAAAATACTAGGAATACGGCTATTGCTCGATTAGAAGGT444                           GlnValThrGluAsnThrArgAsnThrAlaIleAlaArgLeuGluGly                              115120125                                                                     CTAGGAAGAGGCTATAGATCTTACCAGCAGGCTCTTGAAACTTGGTTA492                           LeuGlyArgGlyTyrArgSerTyrGlnGlnAlaLeuGluThrTrpLeu                              130135140                                                                     GATAACCGAAATGATGCAAGATCAAGAAGCATTATTCTTGAGCGCTAT540                           AspAsnArgAsnAspAlaArgSerArgSerIleIleLeuGluArgTyr                              145150155                                                                     GTTGCTTTAGAACTTGACATTACTACTGCTATACCGCTTTTCAGAATA588                           ValAlaLeuGluLeuAspIleThrThrAlaIleProLeuPheArgIle                              160165170                                                                     CGAAATGAAGAAGTTCCATTATTAATGGTATATGCTCAAGCTGCAAAT636                           ArgAsnGluGluValProLeuLeuMetValTyrAlaGlnAlaAlaAsn                              175180185190                                                                  TTACACCTATTATTATTGAGAGACGCATCCCTTTTTGGTAGTGAATGG684                           LeuHisLeuLeuLeuLeuArgAspAlaSerLeuPheGlySerGluTrp                              195200205                                                                     GGGATGGCATCTTCCGATGTTAACCAATATTACCAAGAACAAATCAGA732                           GlyMetAlaSerSerAspValAsnGlnTyrTyrGlnGluGlnIleArg                              210215220                                                                     TATACAGAGGAATATTCTAACCATTGCGTACAATGGTATAATACAGGG780                           TyrThrGluGluTyrSerAsnHisCysValGlnTrpTyrAsnThrGly                              225230235                                                                     CTAAATAACTTAAGAGGGACAAATGCTGAAAGTTGGTTGCGGTATAAT828                           LeuAsnAsnLeuArgGlyThrAsnAlaGluSerTrpLeuArgTyrAsn                              240245250                                                                     CAATTCCGTAGAGACCTAACGTTAGGGGTATTAGATTTAGTAGCCCTA876                           GlnPheArgArgAspLeuThrLeuGlyValLeuAspLeuValAlaLeu                              255260265270                                                                  TTCCCAAGCTATGATACTCGCACTTATCCAATCAATACGAGTGCTCAG924                           PheProSerTyrAspThrArgThrTyrProIleAsnThrSerAlaGln                              275280285                                                                     TTAACAAGAGAAATTTATACAGATCCAATTGGGAGAACAAATGCACCT972                           LeuThrArgGluIleTyrThrAspProIleGlyArgThrAsnAlaPro                              290295300                                                                     TCAGGATTTGCAAGTACGAATTGGTTTAATAATAATGCACCATCGTTT1020                          SerGlyPheAlaSerThrAsnTrpPheAsnAsnAsnAlaProSerPhe                              305310315                                                                     TCTGCCATAGAGGCTGCCATTTTCAGGCCTCCGCATCTACTTGATTTT1068                          SerAlaIleGluAlaAlaIlePheArgProProHisLeuLeuAspPhe                              320325330                                                                     CCAGAACAACTTACAATTTACAGTGCATCAAGCCGTTGGAGTAGCACT1116                          ProGluGlnLeuThrIleTyrSerAlaSerSerArgTrpSerSerThr                              335340345350                                                                  CAACATATGAATTATTGGGTGGGACATAGGCTTAACTTCCGCCCAATA1164                          GlnHisMetAsnTyrTrpValGlyHisArgLeuAsnPheArgProIle                              355360365                                                                     GGAGGGACATTAAATACCTCAACACAAGGACTTACTAATAATACTTCA1212                          GlyGlyThrLeuAsnThrSerThrGlnGlyLeuThrAsnAsnThrSer                              370375380                                                                     ATTAATCCTGTAACATTACAGTTTACGTCTCGAGACGTTTATAGAACA1260                          IleAsnProValThrLeuGlnPheThrSerArgAspValTyrArgThr                              385390395                                                                     GAATCAAATGCAGGGACAAATATACTATTTACTACTCCTGTGAATGGA1308                          GluSerAsnAlaGlyThrAsnIleLeuPheThrThrProValAsnGly                              400405410                                                                     GTACCTTGGGCTAGATTTAATTTTATAAACCCTCAGAATATTTATGAA1356                          ValProTrpAlaArgPheAsnPheIleAsnProGlnAsnIleTyrGlu                              415420425430                                                                  AGAGGCGCCACTACCTACAGTCAACCGTATCAGGGAGTTGGGATTCAA1404                          ArgGlyAlaThrThrTyrSerGlnProTyrGlnGlyValGlyIleGln                              435440445                                                                     TTATTTGATTCAGAAACTGAATTACCACCAGAAACAACAGAACGACCA1452                          LeuPheAspSerGluThrGluLeuProProGluThrThrGluArgPro                              450455460                                                                     AATTATGAATCATATAGTCATAGATTATCTCATATAGGACTAATCATA1500                          AsnTyrGluSerTyrSerHisArgLeuSerHisIleGlyLeuIleIle                              465470475                                                                     GGAAACACTTTGAGAGCACCAGTCTATTCTTGGACGCATCGTAGTGCA1548                          GlyAsnThrLeuArgAlaProValTyrSerTrpThrHisArgSerAla                              480485490                                                                     GATCGTACGAATACGATTGGACCAAATAGAATTACACAAATACCATTG1596                          AspArgThrAsnThrIleGlyProAsnArgIleThrGlnIleProLeu                              495500505510                                                                  GTAAAAGCACTGAATCTTCATTCAGGTGTTACTGTTGTTGGAGGGCCA1644                          ValLysAlaLeuAsnLeuHisSerGlyValThrValValGlyGlyPro                              515520525                                                                     GGATTTACAGGTGGGGATATCCTTCGTAGAACAAATACGGGTACATTT1692                          GlyPheThrGlyGlyAspIleLeuArgArgThrAsnThrGlyThrPhe                              530535540                                                                     GGAGATATACGATTAAATATTAATGTGCCATTATCCCAAAGATATCGC1740                          GlyAspIleArgLeuAsnIleAsnValProLeuSerGlnArgTyrArg                              545550555                                                                     GTAAGGATTCGTTATGCTTCTACTACAGATTTACAATTTTTCACGAGA1788                          ValArgIleArgTyrAlaSerThrThrAspLeuGlnPhePheThrArg                              560565570                                                                     ATTAATGGAACCACTGTTAATATTGGTAATTTCTCAAGAACTATGAAT1836                          IleAsnGlyThrThrValAsnIleGlyAsnPheSerArgThrMetAsn                              575580585590                                                                  AGGGGGGATAATTTAGAATATAGAAGTTTTAGAACTGCAGGATTTAGT1884                          ArgGlyAspAsnLeuGluTyrArgSerPheArgThrAlaGlyPheSer                              595600605                                                                     ACTCCTTTTAATTTTTTAAATGCCCAAAGCACATTCACATTGGGTGCT1932                          ThrProPheAsnPheLeuAsnAlaGlnSerThrPheThrLeuGlyAla                              610615620                                                                     CAGAGTTTTTCAAATCAGGAAGTTTATATAGATAGAGTCGAATTTGTT1980                          GlnSerPheSerAsnGlnGluValTyrIleAspArgValGluPheVal                              625630635                                                                     CCAGCAGAGGTAACATTTGAGGCAGAATATGATTTAGAAAGAGCACAA2028                          ProAlaGluValThrPheGluAlaGluTyrAspLeuGluArgAlaGln                              640645650                                                                     AAGGCGGTGAATGCTCTGTTTACTTCTACAAATCCAAGAAGATTGAAA2076                          LysAlaValAsnAlaLeuPheThrSerThrAsnProArgArgLeuLys                              655660665670                                                                  ACAGATGTGACAGATTATCATATTGACCAAGTGTCCAATATGGTGGCA2124                          ThrAspValThrAspTyrHisIleAspGlnValSerAsnMetValAla                              675680685                                                                     TGTTTATCAGATGAATTTTGCTTGGATGAGAAGCGAGAATTATTTGAG2172                          CysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuPheGlu                              690695700                                                                     AAAGTGAAATATGCGAAGCGACTCAGTGATGAAAGAAACTTACTCCAA2220                          LysValLysTyrAlaLysArgLeuSerAspGluArgAsnLeuLeuGln                              705710715                                                                     GATCCAAACTTCACATTCATCAGTGGGCAATTAAGTTTCGCATCCATC2268                          AspProAsnPheThrPheIleSerGlyGlnLeuSerPheAlaSerIle                              720725730                                                                     GATGGACAATCAAACTTCCCCTCTATTAATGAGCTATCTGAACATGGA2316                          AspGlyGlnSerAsnPheProSerIleAsnGluLeuSerGluHisGly                              735740745750                                                                  TGGTGGGGAAGTGCGAATGTTACCATTCAGGAAGGGAATGACGTATTT2364                          TrpTrpGlySerAlaAsnValThrIleGlnGluGlyAsnAspValPhe                              755760765                                                                     AAAGAGAATTACGTCACACTACCGGGTACTTTTAATGAGTGTTATCCA2412                          LysGluAsnTyrValThrLeuProGlyThrPheAsnGluCysTyrPro                              770775780                                                                     AATTATTTATATCAAAAAATAGGAGAGTCAGAATTAAAAGCTTATACG2460                          AsnTyrLeuTyrGlnLysIleGlyGluSerGluLeuLysAlaTyrThr                              785790795                                                                     CGCTATCAATTAAGAGGGTATATTGAAGATAGTCAAGATCTAGAGATT2508                          ArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIle                              800805810                                                                     TATTTAATTCGTTACAATGCAAAGCATGAAACATTGGATGTTCCAGGT2556                          TyrLeuIleArgTyrAsnAlaLysHisGluThrLeuAspValProGly                              815820825830                                                                  ACCGATTCCCTATGGCCGCTTTCAGTTGAAAGCCCAATCGGAAGGTGC2604                          ThrAspSerLeuTrpProLeuSerValGluSerProIleGlyArgCys                              835840845                                                                     GGAGAACCAAATCGATGCGCACCACATTTTGAATGGAATCCTGATCTA2652                          GlyGluProAsnArgCysAlaProHisPheGluTrpAsnProAspLeu                              850855860                                                                     GATTGTTCCTGCAGAGATGGAGAAAGATGTGCGCATCATTCCCATCAT2700                          AspCysSerCysArgAspGlyGluArgCysAlaHisHisSerHisHis                              865870875                                                                     TTCACTTTGGATATTGATGTTGGGTGCACAGACTTGCATGAGAACCTA2748                          PheThrLeuAspIleAspValGlyCysThrAspLeuHisGluAsnLeu                              880885890                                                                     GGCGTGTGGGTGGTATTCAAGATTAAGACGCAGGAAGGTTATGCAAGA2796                          GlyValTrpValValPheLysIleLysThrGlnGluGlyTyrAlaArg                              895900905910                                                                  TTAGGAAATCTGGAATTTATCGAAGAGAAACCATTAATTGGAGAAGCA2844                          LeuGlyAsnLeuGluPheIleGluGluLysProLeuIleGlyGluAla                              915920925                                                                     CTGTCTCGTGTGAAGAGAGCGGAAAAAAAATGGAGAGACAAACGGGAA2892                          LeuSerArgValLysArgAlaGluLysLysTrpArgAspLysArgGlu                              930935940                                                                     AAACTACAATTGGAAACAAAACGAGTATATACAGAGGCAAAAGAAGCT2940                          LysLeuGlnLeuGluThrLysArgValTyrThrGluAlaLysGluAla                              945950955                                                                     GTGGATGCTTTATTCGTAGATTCTCAATATGATCAATTACAAGCGGAT2988                          ValAspAlaLeuPheValAspSerGlnTyrAspGlnLeuGlnAlaAsp                              960965970                                                                     ACAAACATTGGCATGATTCATGCGGCAGATAAACTTGTTCATCGAATT3036                          ThrAsnIleGlyMetIleHisAlaAlaAspLysLeuValHisArgIle                              975980985990                                                                  CGAGAGGCGTATCTTTCAGAATTACCTGTTATCCCAGGTGTAAATGCG3084                          ArgGluAlaTyrLeuSerGluLeuProValIleProGlyValAsnAla                              99510001005                                                                   GAAATTTTTGAAGAATTAGAAGGTCACATTATCACTGCAATGTCCTTA3132                          GluIlePheGluGluLeuGluGlyHisIleIleThrAlaMetSerLeu                              101010151020                                                                  TACGATGCGAGAAATGTCGTTAAAAATGGTGATTTTAATAATGGATTA3180                          TyrAspAlaArgAsnValValLysAsnGlyAspPheAsnAsnGlyLeu                              102510301035                                                                  ACATGTTGGAATGTAAAAGGGCATGTAGATGTACAACAGAGCCATCAT3228                          ThrCysTrpAsnValLysGlyHisValAspValGlnGlnSerHisHis                              104010451050                                                                  CGTTCTGACCTTGTTATCCCAGAATGGGAAGCAGAAGTGTCACAAGCA3276                          ArgSerAspLeuValIleProGluTrpGluAlaGluValSerGlnAla                              1055106010651070                                                              GTTCGCGTCTGTCCGGGGCGTGGCTATATCCTTCGTGTCACAGCGTAC3324                          ValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyr                              107510801085                                                                  AAAGAGGGATATGGAGAGGGCTGCGTAACGATCCATGAAATCGAGAAC3372                          LysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsn                              109010951100                                                                  AATACAGACGAACTAAAATTTAAAAACTGTGAAGAAGAGGAAGTGTAT3420                          AsnThrAspGluLeuLysPheLysAsnCysGluGluGluGluValTyr                              110511101115                                                                  CCAACGGATACAGGAACGTGTAATGATTATACTGCACACCAAGGTACA3468                          ProThrAspThrGlyThrCysAsnAspTyrThrAlaHisGlnGlyThr                              112011251130                                                                  GCAGCATGTAATTCCCGTAATGCTGGATATGAGGATGCATATGAAGTT3516                          AlaAlaCysAsnSerArgAsnAlaGlyTyrGluAspAlaTyrGluVal                              1135114011451150                                                              GATACTACAGCATCTGTTAATTACAAACCGACTTATGAAGAAGAAACG3564                          AspThrThrAlaSerValAsnTyrLysProThrTyrGluGluGluThr                              115511601165                                                                  TATACAGATGTACGAAGAGATAATCATTGTGAATATGACAGAGGGTAT3612                          TyrThrAspValArgArgAspAsnHisCysGluTyrAspArgGlyTyr                              117011751180                                                                  GTGAATTATCCACCAGTACCAGCTGGTTATGTGACAAAAGAATTAGAA3660                          ValAsnTyrProProValProAlaGlyTyrValThrLysGluLeuGlu                              118511901195                                                                  TACTTCCCAGAAACAGATACAGTATGGATTGAGATTGGAGAAACGGAA3708                          TyrPheProGluThrAspThrValTrpIleGluIleGlyGluThrGlu                              120012051210                                                                  GGAAAGTTTATTGTAGATAGCGTGGAACTACTCCTCATGGAAGAATAGGATCA3763                     GlyLysPheIleValAspSerValGluLeuLeuLeuMetGluGlu                                 121512201225123                                                               CAAGTATAGCAGTTTAATAAATATTAATTAAAATAGTAGTCTAACTTCCGTTCCAATTAA3823              ATAAGTAAATTACAGTTGTAAAAAGAAAACGGACATCACTCTTCAGAGAGCGATGTCCGT3883              TTTTTATATGGTGTGTGCTAATGATAAATGTGCACGAAATTATATTGTCAA3934                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1229 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuThrSerAsnArgLysAsnGluAsnGluIleIleAsnAlaLeuSer                              151015                                                                        IleProThrValSerAsnProSerThrGlnMetAsnLeuSerProAsp                              202530                                                                        AlaArgIleGluAspSerLeuCysValAlaGluValAsnAsnIleAsp                              354045                                                                        ProPheValSerAlaSerThrValGlnThrGlyIleAsnIleAlaGly                              505560                                                                        ArgIleLeuGlyValLeuGlyValProPheAlaGlyGlnLeuAlaSer                              65707580                                                                      PheTyrSerPheLeuValGlyGluLeuTrpProSerGlyArgAspPro                              859095                                                                        TrpGluIlePheLeuGluHisValGluGlnLeuIleArgGlnGlnVal                              100105110                                                                     ThrGluAsnThrArgAsnThrAlaIleAlaArgLeuGluGlyLeuGly                              115120125                                                                     ArgGlyTyrArgSerTyrGlnGlnAlaLeuGluThrTrpLeuAspAsn                              130135140                                                                     ArgAsnAspAlaArgSerArgSerIleIleLeuGluArgTyrValAla                              145150155160                                                                  LeuGluLeuAspIleThrThrAlaIleProLeuPheArgIleArgAsn                              165170175                                                                     GluGluValProLeuLeuMetValTyrAlaGlnAlaAlaAsnLeuHis                              180185190                                                                     LeuLeuLeuLeuArgAspAlaSerLeuPheGlySerGluTrpGlyMet                              195200205                                                                     AlaSerSerAspValAsnGlnTyrTyrGlnGluGlnIleArgTyrThr                              210215220                                                                     GluGluTyrSerAsnHisCysValGlnTrpTyrAsnThrGlyLeuAsn                              225230235240                                                                  AsnLeuArgGlyThrAsnAlaGluSerTrpLeuArgTyrAsnGlnPhe                              245250255                                                                     ArgArgAspLeuThrLeuGlyValLeuAspLeuValAlaLeuPhePro                              260265270                                                                     SerTyrAspThrArgThrTyrProIleAsnThrSerAlaGlnLeuThr                              275280285                                                                     ArgGluIleTyrThrAspProIleGlyArgThrAsnAlaProSerGly                              290295300                                                                     PheAlaSerThrAsnTrpPheAsnAsnAsnAlaProSerPheSerAla                              305310315320                                                                  IleGluAlaAlaIlePheArgProProHisLeuLeuAspPheProGlu                              325330335                                                                     GlnLeuThrIleTyrSerAlaSerSerArgTrpSerSerThrGlnHis                              340345350                                                                     MetAsnTyrTrpValGlyHisArgLeuAsnPheArgProIleGlyGly                              355360365                                                                     ThrLeuAsnThrSerThrGlnGlyLeuThrAsnAsnThrSerIleAsn                              370375380                                                                     ProValThrLeuGlnPheThrSerArgAspValTyrArgThrGluSer                              385390395400                                                                  AsnAlaGlyThrAsnIleLeuPheThrThrProValAsnGlyValPro                              405410415                                                                     TrpAlaArgPheAsnPheIleAsnProGlnAsnIleTyrGluArgGly                              420425430                                                                     AlaThrThrTyrSerGlnProTyrGlnGlyValGlyIleGlnLeuPhe                              435440445                                                                     AspSerGluThrGluLeuProProGluThrThrGluArgProAsnTyr                              450455460                                                                     GluSerTyrSerHisArgLeuSerHisIleGlyLeuIleIleGlyAsn                              465470475480                                                                  ThrLeuArgAlaProValTyrSerTrpThrHisArgSerAlaAspArg                              485490495                                                                     ThrAsnThrIleGlyProAsnArgIleThrGlnIleProLeuValLys                              500505510                                                                     AlaLeuAsnLeuHisSerGlyValThrValValGlyGlyProGlyPhe                              515520525                                                                     ThrGlyGlyAspIleLeuArgArgThrAsnThrGlyThrPheGlyAsp                              530535540                                                                     IleArgLeuAsnIleAsnValProLeuSerGlnArgTyrArgValArg                              545550555560                                                                  IleArgTyrAlaSerThrThrAspLeuGlnPhePheThrArgIleAsn                              565570575                                                                     GlyThrThrValAsnIleGlyAsnPheSerArgThrMetAsnArgGly                              580585590                                                                     AspAsnLeuGluTyrArgSerPheArgThrAlaGlyPheSerThrPro                              595600605                                                                     PheAsnPheLeuAsnAlaGlnSerThrPheThrLeuGlyAlaGlnSer                              610615620                                                                     PheSerAsnGlnGluValTyrIleAspArgValGluPheValProAla                              625630635640                                                                  GluValThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAla                              645650655                                                                     ValAsnAlaLeuPheThrSerThrAsnProArgArgLeuLysThrAsp                              660665670                                                                     ValThrAspTyrHisIleAspGlnValSerAsnMetValAlaCysLeu                              675680685                                                                     SerAspGluPheCysLeuAspGluLysArgGluLeuPheGluLysVal                              690695700                                                                     LysTyrAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspPro                              705710715720                                                                  AsnPheThrPheIleSerGlyGlnLeuSerPheAlaSerIleAspGly                              725730735                                                                     GlnSerAsnPheProSerIleAsnGluLeuSerGluHisGlyTrpTrp                              740745750                                                                     GlySerAlaAsnValThrIleGlnGluGlyAsnAspValPheLysGlu                              755760765                                                                     AsnTyrValThrLeuProGlyThrPheAsnGluCysTyrProAsnTyr                              770775780                                                                     LeuTyrGlnLysIleGlyGluSerGluLeuLysAlaTyrThrArgTyr                              785790795800                                                                  GlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeu                              805810815                                                                     IleArgTyrAsnAlaLysHisGluThrLeuAspValProGlyThrAsp                              820825830                                                                     SerLeuTrpProLeuSerValGluSerProIleGlyArgCysGlyGlu                              835840845                                                                     ProAsnArgCysAlaProHisPheGluTrpAsnProAspLeuAspCys                              850855860                                                                     SerCysArgAspGlyGluArgCysAlaHisHisSerHisHisPheThr                              865870875880                                                                  LeuAspIleAspValGlyCysThrAspLeuHisGluAsnLeuGlyVal                              885890895                                                                     TrpValValPheLysIleLysThrGlnGluGlyTyrAlaArgLeuGly                              900905910                                                                     AsnLeuGluPheIleGluGluLysProLeuIleGlyGluAlaLeuSer                              915920925                                                                     ArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeu                              930935940                                                                     GlnLeuGluThrLysArgValTyrThrGluAlaLysGluAlaValAsp                              945950955960                                                                  AlaLeuPheValAspSerGlnTyrAspGlnLeuGlnAlaAspThrAsn                              965970975                                                                     IleGlyMetIleHisAlaAlaAspLysLeuValHisArgIleArgGlu                              980985990                                                                     AlaTyrLeuSerGluLeuProValIleProGlyValAsnAlaGluIle                              99510001005                                                                   PheGluGluLeuGluGlyHisIleIleThrAlaMetSerLeuTyrAsp                              101010151020                                                                  AlaArgAsnValValLysAsnGlyAspPheAsnAsnGlyLeuThrCys                              1025103010351040                                                              TrpAsnValLysGlyHisValAspValGlnGlnSerHisHisArgSer                              104510501055                                                                  AspLeuValIleProGluTrpGluAlaGluValSerGlnAlaValArg                              106010651070                                                                  ValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGlu                              107510801085                                                                  GlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThr                              109010951100                                                                  AspGluLeuLysPheLysAsnCysGluGluGluGluValTyrProThr                              1105111011151120                                                              AspThrGlyThrCysAsnAspTyrThrAlaHisGlnGlyThrAlaAla                              112511301135                                                                  CysAsnSerArgAsnAlaGlyTyrGluAspAlaTyrGluValAspThr                              114011451150                                                                  ThrAlaSerValAsnTyrLysProThrTyrGluGluGluThrTyrThr                              115511601165                                                                  AspValArgArgAspAsnHisCysGluTyrAspArgGlyTyrValAsn                              117011751180                                                                  TyrProProValProAlaGlyTyrValThrLysGluLeuGluTyrPhe                              1185119011951200                                                              ProGluThrAspThrValTrpIleGluIleGlyGluThrGluGlyLys                              120512101215                                                                  PheIleValAspSerValGluLeuLeuLeuMetGluGlu                                       12201225                                                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AAGTTTCGCATCCATCGATG20                                                        __________________________________________________________________________

We claim:
 1. An isolated lepidopteran-toxic protein having the aminoacid sequence shown in FIG. 1 and listed in SEQ ID NO:2.
 2. An isolatedlepidopteran-toxic protein having the amino acid sequence shown in FIG.2 and listed in SEQ ID NO:4.
 3. An insecticide composition comprisingthe protein of claim 1 and an agriculturally acceptable carrier.
 4. Aninsecticide composition comprising the protein of claim 2 and anagriculturally acceptable carrier.
 5. A method of controllingsusceptible lepidopteran insects comprising applying to a host plant forsuch insects an insect-controlling effective amount of alepidopteran-toxic protein identical to the protein of claim
 1. 6. Themethod of claim 5 wherein the lepidopteran-toxic protein is associatedwith a Bacillus thuringiensis bacterium which has produced such protein.7. The method of claim 5 wherein the lepidopteran insect is Heliothisvirescens, Helicoverpa zea, Lymantria dispar, Ostrinia nubilalis,Pseudoplusia includens, Plutella xylostella, Spodoptera exigua,Spodoptera frugiperda or Trichoplusia ni.
 8. A method of controllingsusceptible lepidopteran insects comprising applying to a host plant forsuch insects an insect-controlling effective amount of alepidopteran-toxic protein identical to the protein of claim
 2. 9. Themethod of claim 8 wherein the lepidopteran-toxic protein is associatedwith a Bacillus thuringiensis bacterium which has produced such protein.10. The method of claim 8 wherein the lepidopteran insect is Lymanatriadispar, Ostrinia nubilalis, Pseudoplusia includens, Plutella xylostella,Spodoptera frugiperda or Trichoplusia ni.